CN111579703A - LC-MS/MS-based method for detecting and analyzing degradation products of bilirubin in biological sample - Google Patents
LC-MS/MS-based method for detecting and analyzing degradation products of bilirubin in biological sample Download PDFInfo
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- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 title claims abstract description 46
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- 238000001514 detection method Methods 0.000 claims abstract description 61
- JJDLLVZMTMEVBY-DAXSKMNVSA-N BOX B Chemical compound CC1=C(C=C)C(=O)N\C1=C/C(N)=O JJDLLVZMTMEVBY-DAXSKMNVSA-N 0.000 claims abstract description 48
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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Abstract
The invention discloses a method for determining the contents of three bilirubin oxidation products, namely BOX A, BOX B and hematic acid, in a biological sample based on high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method utilizes a high performance liquid chromatography-triple quadrupole mass spectrometry combined technology to detect the contents of bilirubin oxidation products BOX A, BOX B and hematonic acid in a sample. The samples were deproteinized, analyzed by LC-MS/MS instrument MRM mode, and quantified by external standard method. The method has the advantages of simplicity, rapidness, low detection limit, good stability, high detection sensitivity and good repeatability, is used for measuring the oxidation product of the bilirubin in the biological sample, can meet the detection requirement of a target object in the biological sample, has high sensitivity and selectivity and strong anti-interference capability, and can be used as an effective tool for qualitative and quantitative analysis of the bilirubin oxidation product.
Description
Technical Field
The invention relates to a detection and analysis method of a biological sample, in particular to a detection and analysis method of a degradation product of bilirubin in cerebrospinal fluid, which is applied to the technical field of detection of a target compound of a biological sample.
Background
Stroke, also known as stroke, is an acute cerebrovascular disease and has the characteristics of high morbidity, high disability rate, high mortality and high recurrence rate. The cerebral apoplexy comprises ischemic stroke and hemorrhagic stroke, wherein the hemorrhagic stroke is caused by rupture and bleeding of cerebral artery blood vessels, and blood overflows into brain tissues to cause the damage of the brain tissues, thereby generating local dysfunction of the brain. Subarachnoid hemorrhage is a form of hemorrhagic stroke with higher mortality than the average mortality of stroke.
Studies have shown that lysis of erythrocytes following subarachnoid hemorrhage is a major cause of patient recovery. The red blood cells in the blood are cracked to release hemoglobin, the heme in the hemoglobin is further degraded into biliverdin, and the biliverdin is reduced into bilirubin under the action of biliverdin reductase. Bilirubin acts as a reducing agent, scavenging active oxygen, and as a protective agent in the event of oxidative stress in cells due to its antioxidant properties. Research shows that cerebral hemorrhage can cause strong oxidative stress, release active oxygen, bilirubin is used as a reducing agent, and the cerebral hemorrhage can cause the strong oxidative stress when encountering H2O2Post-oxidation to small molecule compounds, in which BOX A
(2-(3-ethenyl-1,5-dihydro-4-methyl-5-oxo-2H-pyrrol-2-ylidene)acetamide，bilirubin oxidizedA，CAS#329314-76-3)、BOX B
(2- (4-ethyl-1, 5-dihydro-3-methyl-5-oxo-2H-pyrro-2-ylene) acetamide, bilirubin oxidized B, CAS #329314-75-2) and humic acid (2,5-dihydro-4-methyl-2, 5-oxo-1H-pyrro-3-propanoic acid, CAS #487-65-0) are reported as H2O2Oxidation of the degradation products of bilirubin. Experiments prove that the BOXA and the BOX B can cause severe and persistent vasospasm of rat cerebral vessels, and have important physiological significance; hematinic acid is the earliest reported oxidation product of bilirubin and no biological studies have been reported for this compound. To study the contribution of bilirubin oxidation products to cerebral hemorrhage complications, the most direct and effective method is to measure the concentration of each compound in clinical samples and to evaluate the relationship between the concentration of each compound and the disease. But toSo far, a detection method with high sensitivity and high selectivity is still lacking.
Disclosure of Invention
In order to solve the problems of the prior art, the invention aims to overcome the defects in the prior art and provide a detection and analysis method for bilirubin degradation products in a biological sample based on LC-MS/MS.
In order to achieve the purpose of the invention, the invention adopts the following technical scheme:
a detection and analysis method for degradation products of bilirubin in a biological sample based on LC-MS/MS comprises the steps of selecting liquid phase conditions, setting mass spectrum conditions, preparing standard solution, optimizing an analysis method, drawing a standard curve, pretreating the sample and qualitatively and quantitatively detecting products in cerebrospinal fluid; the detection and analysis method can be used for rapidly and accurately determining the BOX A compound, the BOX B compound and the biochemical acid in cerebrospinal fluid, and meanwhile, the detection and analysis method is also used for measuring information of cell lysate and blood samples.
As a preferred technical scheme of the invention, the method for detecting and analyzing the degradation product of bilirubin in a biological sample based on LC-MS/MS comprises the following steps:
s1: selecting a liquid chromatographic column and setting mass spectrum conditions and parameters:
a. setting high performance liquid chromatography conditions:
a chromatographic column: agilent Poroshell EC-C18 column, 2.1X 100mm,2.7 μm; column temperature: 30 ℃; setting a mobile phase and a mobile phase gradient, wherein the ratio of the water phase A: water and 0.1% (v/v) formic acid, organic phase B: methanol and 0.1% (v/v) formic acid, methanol, acetonitrile or acetonitrile and 0.1% (v/v) formic acid, the flow rate is 0.3mL/min, and the sample injection volume of LC-MS/MS is set to be 1-20 mu L;
b. setting mass spectrum conditions:
selecting an electrospray ionization source (ESI), and selecting an appropriate ion scanning mode according to the response of the compound;
c. setting a mass spectrum qualitative and quantitative detection mode:
a multiple reaction monitoring method (MRM) for setting multiple reaction monitoring mode parameters;
s2: preparation of standard solutions:
a. respectively weighing 2mg of BOX A, BOX B and HEMATIC acid, and dissolving in 1.1mL of water to obtain 10mM mother liquor;
b. diluting with water to obtain hemitic acid solution with molar concentration of 500 μ M, and BOXA and BOX B with molar concentration of 10 μ M respectively, storing in a refrigerator at 4 deg.C in dark place for use;
s3: optimizing an instrument analysis method by using a standard solution to obtain an optimized LC-MS/MS quantitative analysis method, wherein the instrument is a liquid chromatogram-tandem triple quadrupole mass spectrometer;
s4: drawing a standard curve:
diluting the hemamatic acid standard mother liquor prepared in the step S2 in proportion to prepare hemamatic acid standard solutions with molar concentrations of 1,5, 10, 50, 100, 150 and 200 mu M respectively, diluting BOX A standard mother liquor and BOX B standard mother liquor in proportion to prepare BOX A standard solutions and BOX B standard solutions with molar concentrations of 5, 10, 50, 100, 200, 400, 600, 800 and 1000nM respectively, and injecting the sample volume of 1 mu L; drawing a standard curve by using the peak area to the molar quantity of the target object, wherein the peak area corresponds to a ordinate y parameter, and the molar quantity of the target object corresponds to an abscissa x parameter;
s5: sample pretreatment:
adding 9 times volume of glacial ethanol into cerebrospinal fluid sample, standing on ice for 30-60min, centrifuging at 15000rpm for 15-45min, and collecting supernatant; concentrating the supernatant with a centrifugal concentrator, and redissolving; filtering with 0.22 μm filter membrane, and performing on-machine test on the filtrate;
s6: quantifying by an external standard method:
after the sample is pretreated in the step S5, the peak area of each compound in cerebrospinal fluid is calculated according to the drawn chromatogram, the concentrations of BOX A, BOX B and heliotic acid in the sample solution are calculated by obtaining the standard curve in the step S4, the linear relation between BOX A and BOX B is in the range of 5-1000fmol, and heliotic acid is in the range of 1-200pmol, the method detection Limit (LOD), the quantitative detection Limit (LOQ) and the detection repeatability information of the calculating instrument of the instrument are calculated, and the precision in the day and the time of the day are calculated at the same time.
As a preferred technical scheme of the invention, in the step a of the step S1, the selected LC-MS/MS instruments are Agilent1290 high performance liquid chromatography system and AB SCIEX 4500 triple quadrupole mass spectrometry system.
As a preferred embodiment of the present invention, in step b of step S1, the mass spectrometry conditions are set as follows:
selecting an electrospray ion source (ESI), scanning a BOX A and a BOX B in a positive ion mode and scanning a hemigenic acid in a negative ion mode according to ion response, respectively injecting 300 mu g/L of standard stock solution into the ion source at a flow rate of 7-20 mu L/min by adopting a semi-automatic sample injection mode, selecting a corresponding parent ion peak, performing secondary mass spectrometry on the child ions of the parent ion peak to obtain fragment ion information, and establishing an MRM scanning method and parameters.
As a preferred technical scheme of the invention, the standard stock solution directly injected into the ion source is prepared by using a mixed solvent of 50% (v/v) methanol and 50% (v/v) water.
As a preferred technical scheme, when an MRM scanning method and parameters are established based on a detection and analysis method of the degradation products of bilirubin in cerebrospinal fluid by LC-MS/MS, the parameters of monitoring ions of each compound are set to comprise parent ions, ionic ions, declustering voltage (DP) and Collision Energy (CE).
As a preferred embodiment of the present invention, in step c of step S1, the other conditions of the MRM detection mode analysis of LC-MS/MS are: the ion source temperature is 500 ℃, the ion spray voltage is 5500V, the collision gas medium, the interface heating is on, the collision chamber inlet voltage (EP) is 10V, the collision chamber outlet voltage (CXP) is 11V, the gas curtain pressure is 40psi, the spray gas pressure is 50psi, and the auxiliary heating gas pressure is 50 psi.
In a preferred embodiment of the present invention, in the step S5, the centrifugation time is 20 to 60 min.
In a preferred embodiment of the present invention, in the step S6, the detection Limit (LOD) of the device is calculated as S/N being 3, the quantitative detection Limit (LOQ) of the device is calculated as S/N being 10, and the reproducibility of detection is represented as RSD% of the peak area of the standard.
Further, the optimization of the instrument analysis method in the step S3 includes optimization of liquid chromatography conditions, optimization of mass spectrometry detection conditions and optimization of mass spectrometry parameters, so that the mass spectrometry can be accurately determined, and the quantitative sensitivity is optimal.
Further, the amount of cerebrospinal fluid treated in the above step S5 is not limited, and the re-lysing volume is not limited.
Further, the biological sample processed in the above step S5 may be other biological samples besides cerebrospinal fluid.
Further, the results were obtained by continuously measuring 1. mu.M of BOX A and BOX B solutions and 200. mu.M of humic acid solution for 3 days at the intra-day and inter-day precision of the above step S6, 3 times per day.
Compared with the prior art, the invention has the following obvious and prominent substantive characteristics and remarkable advantages:
1. according to the invention, the standard substances of the three compounds are utilized to optimize the instrument analysis method, including optimization of liquid chromatography conditions, optimization of mass spectrum detection conditions and optimization of mass spectrum parameters, so that the mass spectrum can be accurately determined, and the quantitative sensitivity reaches the optimum;
2. the method adopts LC-MS/MS, and uses a high performance liquid chromatography-tandem triple quadrupole mass spectrometer to establish a qualitative and quantitative method of three oxidation products of bilirubin in cerebrospinal fluid, and the method has the advantages of simplicity, rapidness, small sample amount, low detection limit, good repeatability and high sensitivity;
3. the method is simple and easy to implement, low in cost and suitable for popularization and application.
Drawings
FIG. 1 is a chromatogram of a standard solution of a hemamatic acid according to example two of the present invention.
Fig. 2 is a chromatogram of a BOX a standard solution of example two of the present invention.
Fig. 3 is a chromatogram of a BOX B standard solution of example two of the present invention.
FIG. 4 is a chromatogram for detection of negative ion pattern in cerebrospinal fluid (detected hematonic) in example two of the present invention.
FIG. 5 is a chromatogram for positive ion mode detection in cerebrospinal fluid (BOX A and BOXB detected) according to example two of the present invention.
Detailed Description
The technical solution in the embodiments of the present invention will be clearly and completely described below. It is to be understood that the embodiments described are only some of the embodiments of the invention, and not all of them. Based on the embodiments of the present invention, it is within the scope of the present invention to use this method in the art to detect BOX A, BOX B and hemanitic acid in cerebrospinal fluid, blood and other biological samples.
The above-described scheme is further illustrated below with reference to specific embodiments, which are detailed below:
the first embodiment is as follows:
in this embodiment, a method for detecting and analyzing degradation products of bilirubin in a biological sample based on LC-MS/MS is characterized in that the detecting and analyzing step includes selecting a liquid phase condition, setting a mass spectrometry condition, preparing a standard solution, optimizing an analysis method, drawing a standard curve, pre-treating the sample, and qualitatively and quantitatively detecting products in cerebrospinal fluid; the detection and analysis method can be used for rapidly and accurately determining the BOX A compound, the BOX B compound and the biochemical acid in cerebrospinal fluid, and meanwhile, the detection and analysis method is also used for measuring information of cell lysate and blood samples.
In the method, LC-MS/MS is used for establishing a method for measuring the content of BOX A, BOX B and hemanitic acid in cerebrospinal fluid. The LC-MS/MS has high sensitivity, high selectivity and strong anti-interference capability, and can be used as an effective tool for qualitative and quantitative analysis of bilirubin oxidation products.
Example two:
this embodiment is substantially the same as the first embodiment, and is characterized in that:
in this example, the instrument was selected for the Agilent1290 hplc system and the AB SCIEX 4500 triple quadrupole mass spectrometry system. A detection and analysis method for degradation products of bilirubin in cerebrospinal fluid based on LC-MS/MS comprises the following steps:
s1: selecting a liquid chromatographic column and setting mass spectrum conditions and parameters:
a. setting high performance liquid chromatography conditions:
a chromatographic column: agilent Poroshell EC-C18 column, 2.1X 100mm,2.7 μm; column temperature: 30 ℃; setting a mobile phase and a mobile phase gradient, wherein the ratio of the water phase A: water and 0.1% (v/v) formic acid, organic phase B: methanol and 0.1% (v/v) formic acid, the flow rate is 0.3mL/min, and the sample injection volume of LC-MS/MS is set to be 1-20 muL;
TABLE 1 gradient parameters of mobile phase
b. Setting mass spectrum conditions, and setting a mass spectrum qualitative and quantitative detection mode:
selecting an electrospray ionization source (ESI), and selecting an appropriate ion scanning mode according to the response of the compound; setting a multi-reaction monitoring mode parameter by adopting a multi-reaction monitoring method (MRM); the method comprises the following specific steps:
selecting an electrospray ion source, scanning a BOX A and a BOX B in a positive ion mode, scanning a chiral acid in a negative ion mode, and respectively injecting 300 mu g/L of standard stock solution into the ion source at a flow rate of 7 mu L/min in a semi-automatic sample injection mode; selecting a corresponding parent ion peak, performing secondary mass spectrometry on the child ions of the parent ion peak to obtain fragment ion information, and establishing an MRM mass spectrometry detection method of the three compounds;
TABLE 2 MRM Mass Spectrometry detection parameters for three compounds
In the mass spectrometry process in the above scheme, the ion source temperature is 500 ℃, the ion spray voltage is 5500V, the collision gas medium, the interface heating is on, the EP is 10V, the CXP is 11V, the gas curtain pressure is 40psi, the spray gas pressure is 50psi, and the auxiliary heating gas pressure is 50 psi;
s2: preparation of standard solutions:
a. respectively weighing 2mg of BOX A, BOX B and HEMATIC acid, and dissolving in 1.1mL of water to obtain 10mM mother liquor;
b. diluting with water to obtain hemitic acid solution with molar concentration of 500 μ M, and BOXA and BOX B with molar concentration of 10 μ M respectively, storing in a refrigerator at 4 deg.C in dark place for use;
s3: optimizing an instrument analysis method by using a standard solution to obtain an optimized LC-MS/MS quantitative analysis method, wherein the instrument is a liquid chromatogram-tandem triple quadrupole mass spectrometer;
s4: drawing a standard curve:
diluting the hemamatic acid standard mother liquor prepared in the step S2 in proportion to prepare hemamatic acid standard solutions with molar concentrations of 1,5, 10, 50, 100, 150 and 200 mu M respectively, diluting BOX A standard mother liquor and BOX B standard mother liquor in proportion to prepare BOX A standard solutions and BOX B standard solutions with molar concentrations of 5, 10, 50, 100, 200, 400, 600, 800 and 1000nM respectively, and injecting the sample volume of 1 mu L; drawing a standard curve by using the peak area to the molar quantity of the target object, wherein the peak area corresponds to a ordinate y parameter, and the molar quantity of the target object corresponds to an abscissa x parameter; the obtained standard curve is shown in the results in table 3.
TABLE 3 LC-MS/MS quantitative analysis method results of three compounds
S5: sample pretreatment:
taking 5mL of 6 cerebrospinal fluid samples, respectively adding 9 times of ice-cold ethanol, placing on ice for 30min, centrifuging at 15000rpm for 30min, taking supernate, centrifuging and concentrating to 5mL, finding that protein exists, adding 9 times of ice-cold ethanol, placing on ice for 40min, centrifuging at 15000rpm for 30min, taking supernate, centrifuging and concentrating to dryness, adding 50 mu L of water for redissolving, and waiting for LC-MS/MS determination;
s6: quantifying by an external standard method:
after the sample is pretreated in the step S5, the peak area of each compound in cerebrospinal fluid is calculated according to the drawn chromatogram, the concentrations of BOX A, BOX B and kinetic acid in the sample solution are calculated by obtaining the standard curve in the step S4, and 6 samples are detected by the established MRM detection method of BOX A, BOX B and kinetic acid on the LC-MS/MS instrument. The obtained peak area is converted by a corresponding working curve and multiplied by the concentration multiple (100 times) to obtain the concentration of each compound in 6 samples; the LC-MS/MS spectra of one of the samples are shown in FIGS. 4 and 5; the results of the 6 samples are summarized in Table 4;
TABLE 4 concentration of each product measured in cerebrospinal fluid
Experimental test analysis:
for the energy-saving test analysis of the technical effect of the method of the embodiment, fig. 1 is a chromatogram of the biochemical standard solution of the second embodiment of the invention. Fig. 2 is a chromatogram of a BOX a standard solution of example two of the present invention. Fig. 3 is a chromatogram of a BOX B standard solution of example two of the present invention. The experimental verification specifically comprises the following steps:
1. selection of mobile phase: in order to optimize the mobile phase conditions, ESI is adopted for BOX A and BOX B in the experiment+Mode, using ESI for the textual acid-Mode for the invention comparison of the separation effects of BOX a, BOX B and chromatographic column was carried out using acetonitrile-water, acetonitrile (containing 0.1% formic acid) -water (containing 0.1% formic acid), methanol-water and methanol (containing 0.1% formic acid) -water (containing 0.1% formic acid) as mobile phases, respectively, and as a result, it was found that the mass spectral response and the degree of separation of the target peak were better than those of acetonitrile-water solution when methanol-water was used as the mobile phase, and addition of formic acid maintained the peak stability and also the pH stability, contributing to extension of the life of the column, so methanol (containing 0.1% formic acid) -water (containing 0.1% formic acid) was selected as the mobile phase.
2. Selection of elution procedure: the elution program was compared for different starting concentrations and different slopes and finally the elution program shown in table 1 was chosen.
The method of the embodiment utilizes a high performance liquid chromatography-triple quadrupole mass spectrometry combined technology to detect the contents of bilirubin oxidation products BOX A, BOX B and hematinic acid in a sample. The samples were deproteinized, analyzed by LC-MS/MS instrument MRM mode, and quantified by external standard method. The method has the advantages of simplicity, rapidness, low detection limit, good stability, high detection sensitivity and good repeatability, is used for measuring oxidation products BOX A, BOX B and hematonic acid of bilirubin in cerebrospinal fluid, and can meet the detection requirement of a target object in the cerebrospinal fluid. In this example, LC-MS/MS was used to establish a method for determining the content of BOX A, BOX B and hemanitic acid in cerebrospinal fluid. The LC-MS/MS has high sensitivity, high selectivity and strong anti-interference capability, and can be used as an effective tool for qualitative and quantitative analysis of bilirubin oxidation products.
Example three:
the detection and analysis method based on the LC-MS/MS for the degradation products of bilirubin in the biological sample can be used for cerebrospinal fluid detection, and can also be used for detecting the concentrations of BOX A, BOX B and hematonic acid in other biological samples such as blood, cell lysate and the like.
Bilirubin oxidation products can cause cerebral vasospasm, suggesting that they may be physiologically toxic. BOX a, BOX B and hemoglobin acid may be generated when the blood contains a large amount of red blood cells, and other diseases may be caused when the blood content is too high.
The detection method for detecting the concentration of each compound in the blood sample specifically comprises the following steps:
s1: selecting a liquid chromatographic column and setting mass spectrum conditions and parameters:
a. high performance liquid chromatography conditions: a chromatographic column: agilent Poroshell EC-C18 column, 2.1X 100mm,2.7 μm; column temperature: 30 ℃; setting a mobile phase and a mobile phase gradient, wherein the ratio of the water phase A: water and 0.1% (v/v) formic acid, organic phase B: methanol and 0.1% (v/v) formic acid at a flow rate of 0.3 mL/min;
b. mass spectrum conditions: selecting an electrospray ion source, wherein BOX A and BOX B are scanned in a positive ion mode, and a biochemical is scanned in a negative ion mode, and respectively injecting 300 mu g/L of standard stock solution into the ion source at a flow rate of 7 mu L/min by adopting a semi-automatic sample injection mode; selecting a corresponding parent ion peak, and performing secondary mass spectrometry on the child ions of the parent ion peak to obtain fragment ion information;
c. setting a mass spectrum qualitative and quantitative detection mode: MRM mode, setting parameters: the ion source temperature is 500 ℃, the ion spray voltage is 5500V, the collision gas medium, the interface heating is on, the EP is 10V, the CXP is 11V, the air curtain pressure is 40psi, the spray gas pressure is 50psi, and the auxiliary heating gas pressure is 50 psi;
s2: preparation of standard solutions:
a. respectively weighing 2mg of BOX A, BOX B and HEMATIC acid, and dissolving in 1.1mL of water to obtain 10mM mother liquor;
b. diluting with water to obtain a heminic acid solution with a molar concentration of 500. mu.M (91.5. mu.g/mL), 10. mu.M (1.79. mu.g/mL) BOX A and BOX B, and storing in a refrigerator at 4 ℃ in the dark for later use;
s3: optimizing an instrument analysis method by using a standard solution to obtain an optimized LC-MS/MS quantitative analysis method, wherein the instrument is a liquid chromatogram-tandem triple quadrupole mass spectrometer;
s4: drawing a standard curve:
diluting the hemitic acid standard mother liquor in S2 in proportion to prepare hemitic acid standard solutions with molar concentrations of 1,5, 10, 50, 100, 150 and 200 mu M, respectively diluting BOX A standard mother liquor and BOX B standard mother liquor in proportion to prepare BOX A standard solutions and BOX B standard solutions with molar concentrations of 5, 10, 50, 100, 200, 400, 600, 800 and 1000nM, and injecting the sample volume of 1 mu L;
drawing a standard curve by the peak area (y) to the molar quantity (x) of the target;
s5: sample pretreatment:
collecting serum from fresh 4mL blood sample, adding ice-cold ethanol 9 times the volume of the blood sample, standing on ice for 30min, centrifuging at 15000rpm for 30min, collecting supernatant, and repeating the above steps until no protein precipitate is in the supernatant;
concentrating the supernatant with a centrifugal concentrator to dryness, and redissolving with 40 μ L of water;
filtering with 0.22 μm filter membrane, and performing on-machine test on the filtrate;
s6: quantifying by an external standard method: after the analysis of step S5, the peak area of each compound in the serum is calculated according to the drawn chromatogram, the concentration of BOX A, BOX B and chromatographic acid in the sample solution is calculated by the standard curve described in step S4, BOX A and BOX B are in the range of 5-1000fmol, the chromatographic acid is in good linear relation in the range of 1-200pmol, the LOD of the instrument is calculated by S/N-3, the LOQ of the instrument is calculated by S/N-10, and the daily and diurnal precision is obtained by continuously measuring 3 times per day by using 1 μ M BOX A and BOX B solution and 200 μ M chromatographic acid solution.
The method is a method for measuring the contents of three bilirubin oxidation products, namely BOX A, BOX B and hematonic acid, in a biological sample based on high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method utilizes a high performance liquid chromatography-triple quadrupole mass spectrometry combined technology to detect the contents of bilirubin oxidation products BOX A, BOX B and hematonic acid in a sample. The samples were deproteinized, analyzed by LC-MS/MS instrument MRM mode, and quantified by external standard method. The method has the advantages of simplicity, rapidness, low detection limit, good stability, high detection sensitivity and good repeatability, is used for measuring oxidation products BOX A, BOX B and hematonic acid of bilirubin in a biological sample, and can meet the detection requirement of a target object in cerebrospinal fluid.
While the embodiments of the present invention have been described with reference to the accompanying drawings, the present invention is not limited to the above embodiments, and various changes, modifications, substitutions, combinations or simplifications made according to the spirit and principle of the present invention should be made in an equivalent manner without departing from the technical principles and inventive concept of the method for detecting and analyzing degradation products of bilirubin in biological samples by LC-MS/MS.
Claims (9)
1. A detection and analysis method for degradation products of bilirubin in a biological sample based on LC-MS/MS is characterized in that the detection and analysis step comprises the steps of selecting liquid phase conditions, setting mass spectrum conditions, preparing standard solution, optimizing the analysis method, drawing a standard curve, pretreating the sample and qualitatively and quantitatively detecting products in cerebrospinal fluid; the detection and analysis method can be used for rapidly and accurately determining the BOX A compound, the BOX B compound and the biochemical acid in cerebrospinal fluid, and meanwhile, the detection and analysis method is also used for measuring information of cell lysate and blood samples.
2. The LC-MS/MS-based assay for the detection of degradation products of bilirubin in a biological sample as described in claim 1 wherein: the method comprises the following steps:
s1: selecting a liquid chromatographic column and setting mass spectrum conditions and parameters:
a. setting high performance liquid chromatography conditions:
a chromatographic column: agilent Poroshell EC-C18 column, 2.1X 100mm,2.7 μm; column temperature: 30 ℃; setting a mobile phase and a mobile phase gradient, wherein the ratio of the water phase A: water and 0.1% (v/v) formic acid, organic phase B: methanol and 0.1% (v/v) formic acid, methanol, acetonitrile or acetonitrile and 0.1% (v/v) formic acid, the flow rate is 0.3mL/min, and the sample injection volume of LC-MS/MS is set to be 1-20 mu L;
b. setting mass spectrum conditions:
selecting an electrospray ionization source (ESI), and selecting an appropriate ion scanning mode according to the response of the compound;
c. setting a mass spectrum qualitative and quantitative detection mode:
a multiple reaction monitoring method (MRM) for setting multiple reaction monitoring mode parameters;
s2: preparation of standard solutions:
a. respectively weighing 2mg of BOX A, BOX B and HEMATIC acid, and dissolving in 1.1mL of water to obtain 10mM mother liquor;
b. diluting with water to obtain hemitic acid solution with molar concentration of 500 μ M, and storing in a refrigerator at 4 deg.C in dark condition to obtain BOX A and BOX B with molar concentration of 10 μ M;
s3: optimizing an instrument analysis method by using a standard solution to obtain an optimized LC-MS/MS quantitative analysis method, wherein the instrument is a liquid chromatogram-tandem triple quadrupole mass spectrometer;
s4: drawing a standard curve:
diluting the hemamatic acid standard mother liquor prepared in the step S2 in proportion to prepare hemamatic acid standard solutions with molar concentrations of 1,5, 10, 50, 100, 150 and 200 mu M respectively, diluting BOX A standard mother liquor and BOX B standard mother liquor in proportion to prepare BOX A standard solutions and BOX B standard solutions with molar concentrations of 5, 10, 50, 100, 200, 400, 600, 800 and 1000nM respectively, and injecting the sample volume of 1 mu L; drawing a standard curve by using the peak area to the molar quantity of the target object, wherein the peak area corresponds to a ordinate y parameter, and the molar quantity of the target object corresponds to an abscissa x parameter;
s5: sample pretreatment:
adding 9 times volume of glacial ethanol into cerebrospinal fluid sample, standing on ice for 30-60min, centrifuging at 15000rpm for 15-45min, and collecting supernatant; concentrating the supernatant with a centrifugal concentrator, and redissolving; filtering with 0.22 μm filter membrane, and performing on-machine test on the filtrate;
s6: quantifying by an external standard method:
after the sample is pretreated in the step S5, the peak area of each compound in cerebrospinal fluid is calculated according to the drawn chromatogram, the concentrations of BOX A, BOX B and heliotic acid in the sample solution are calculated by obtaining the standard curve in the step S4, the linear relation between BOX A and BOX B is in the range of 5-1000fmol, and heliotic acid is in the range of 1-200pmol, the method detection Limit (LOD), the quantitative detection Limit (LOQ) and the detection repeatability information of the calculating instrument of the instrument are calculated, and the precision in the day and the time of the day are calculated at the same time.
3. The LC-MS/MS-based assay for the detection of degradation products of bilirubin in a biological sample as described in claim 2 wherein: in step a of step S1, the LC-MS/MS instrument selected is Agilent1290 hplc system and AB SCIEX 4500 triple quadrupole mass spectrometry system.
4. The LC-MS/MS-based assay for the detection of degradation products of bilirubin in a biological sample as described in claim 2 wherein: in step b of step S1, the mass spectrometry conditions are set as follows:
selecting an electrospray ion source (ESI), scanning a BOX A and a BOX B in a positive ion mode and scanning a hemigenic acid in a negative ion mode according to ion response, respectively injecting 300 mu g/L of standard stock solution into the ion source at a flow rate of 7-20 mu L/min by adopting a semi-automatic sample injection mode, selecting a corresponding parent ion peak, performing secondary mass spectrometry on the child ions of the parent ion peak to obtain fragment ion information, and establishing an MRM scanning method and parameters.
5. The LC-MS/MS-based assay for the detection of degradation products of bilirubin in a biological sample as described in claim 4 wherein: standard stock solutions for direct injection into ion sources were prepared using a mixture of 50% (v/v) methanol and 50% (v/v) water.
6. The LC-MS/MS-based assay for the detection of degradation products of bilirubin in a biological sample as described in claim 4 wherein: when the MRM scanning method and parameters are established, the parameters of the monitored ions of each compound are set to comprise parent ions, ionic ions, cluster removing voltage (DP) and Collision Energy (CE).
7. The method for detecting and analyzing degradation products of bilirubin in a biological sample based on LC-MS/MS as claimed in claim 2, wherein in step c of step S1, the other conditions of the MRM detection mode analysis of LC-MS/MS are as follows: the ion source temperature is 500 ℃, the ion spray voltage is 5500V, the collision gas medium, the interface heating is on, the collision chamber inlet voltage (EP) is 10V, the collision chamber outlet voltage (CXP) is 11V, the gas curtain pressure is 40psi, the spray gas pressure is 50psi, and the auxiliary heating gas pressure is 50 psi.
8. The LC-MS/MS-based detection and analysis method for degradation products of bilirubin in a biological sample as described in claim 2, wherein in said S5 step, the centrifugation treatment time is 20-60 min.
9. The LC-MS/MS-based detection and analysis method for degradation products of bilirubin in a biological sample as described in claim 2, wherein in said S6 step, the detection Limit (LOD) of the instrument is calculated as S/N-3, S/N-10 is used as the quantitative detection Limit (LOQ) of the instrument, and the reproducibility of detection is expressed as RSD% of the area of the standard peak.
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